Bottom Line:
Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ.Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model.Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF.

ABSTRACTWe evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.

Mentions:
Upregulation of BAFF expression by Poly (I:C)- and IFN-γ-activated RAFLS, but not upon TLR2 or TLR4 activation, has been previously reported [13], [22]. To gain more insights into the physiopathological consequences of this observation, we first compared BAFF expression in FLS isolated from healthy donors or RA patients. As shown in figure 1A and B, we observed that IFN-γ-dependent BAFF expression reaches maximum levels in RAFLS, whereas FLS isolated from healthy donors (NFLS) exhibit reduced cytokine expression at both mRNA and protein levels. Of note, Poly (I:C) stimulation induced very limited BAFF expression and secretion by NFLS, while RAFLS appeared extremely responsive to this stimulus. Next, we tested whether this difference between a healthy and a pathological (inflammatory) state could also be observed in another fibroblastic cell type and for this, we chose Human Dermal Fibroblasts (HDF) isolated from skin biopsies harvested from healthy donors (NHDF) or from patients suffering from systemic sclerosis (SScHDF). IFN-γ stimulation (1 and 5 ng/mL) resulted in a comparable increased expression of BAFF transcripts (figure 1C) and cytokine secretion (figure 1D) by NHDF or SScHDF. Interestingly, up-regulation of BAFF transcripts and protein release in response to TLR3 triggering by Poly (I:C) was only detectable in SScHDF and not from healthy individuals. We also investigated here the ability of Bacterial LipoProteins (BLP, Pam3CSK4) or LipoPolysaccharide (LPS), which are respectively ligands for TLR2 and 4, to stimulate BAFF synthesis by NHDF and SScHDF. As seen in figure 1C and D, these PAMPs (Pathogen Associated Molecular Pattern) are not activators of BAFF transcription.

Mentions:
Upregulation of BAFF expression by Poly (I:C)- and IFN-γ-activated RAFLS, but not upon TLR2 or TLR4 activation, has been previously reported [13], [22]. To gain more insights into the physiopathological consequences of this observation, we first compared BAFF expression in FLS isolated from healthy donors or RA patients. As shown in figure 1A and B, we observed that IFN-γ-dependent BAFF expression reaches maximum levels in RAFLS, whereas FLS isolated from healthy donors (NFLS) exhibit reduced cytokine expression at both mRNA and protein levels. Of note, Poly (I:C) stimulation induced very limited BAFF expression and secretion by NFLS, while RAFLS appeared extremely responsive to this stimulus. Next, we tested whether this difference between a healthy and a pathological (inflammatory) state could also be observed in another fibroblastic cell type and for this, we chose Human Dermal Fibroblasts (HDF) isolated from skin biopsies harvested from healthy donors (NHDF) or from patients suffering from systemic sclerosis (SScHDF). IFN-γ stimulation (1 and 5 ng/mL) resulted in a comparable increased expression of BAFF transcripts (figure 1C) and cytokine secretion (figure 1D) by NHDF or SScHDF. Interestingly, up-regulation of BAFF transcripts and protein release in response to TLR3 triggering by Poly (I:C) was only detectable in SScHDF and not from healthy individuals. We also investigated here the ability of Bacterial LipoProteins (BLP, Pam3CSK4) or LipoPolysaccharide (LPS), which are respectively ligands for TLR2 and 4, to stimulate BAFF synthesis by NHDF and SScHDF. As seen in figure 1C and D, these PAMPs (Pathogen Associated Molecular Pattern) are not activators of BAFF transcription.

Bottom Line:
Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ.Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model.Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF.

ABSTRACTWe evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.